Resources
Plasmid Files

pMSCV-LTR-dCas9-p65AD-BFP

Retroviral CRISPR activation vector for expressing TagBFP plus catalytically inactive dCas9 fused to the RelA (p65) transcriptional activation domain.

To see this sequence with restriction sites, features, and translations, please download
 SnapGene or the free  SnapGene Viewer.

pMSCV-LTR-dCas9-p65AD-BFP.dna
Map and Sequence File:    Download    Open   
Sequence Author:  Qi & Weissman Labs / Addgene #46913
Download Free Trial Get SnapGene Viewer


SgrAI (11,809) NdeI (11,437) NsiI (8303) SacII (7864) RsrII (7766) BsiWI (7706) BfuAI - BspMI (7639) AgeI (7240) EcoRI (7120) PaeR7I - XhoI (7108) BamHI (7096) PshAI (7024) BbsI (6692) BglII (1410) NcoI (1421) PflMI (1722) SgrDI (1789) BsaBI * (2240) PsiI (2540) MluI (5256) pMSCV-LTR-dCas9-p65AD-BFP 11,987 bp
SgrAI  (11,809)
1 site
C R C C G G Y G G Y G G C C R C

Efficient cleavage requires at least two copies of the SgrAI recognition sequence.
NdeI  (11,437)
1 site
C A T A T G G T A T A C

Prolonged incubation with NdeI may lead to removal of additional nucleotides.
NsiI  (8303)
1 site
A T G C A T T A C G T A
SacII  (7864)
1 site
C C G C G G G G C G C C

Efficient cleavage requires at least two copies of the SacII recognition sequence.
RsrII  (7766)
1 site
C G G W C C G G C C W G G C

Efficient cleavage requires at least two copies of the RsrII recognition sequence.
Sticky ends from different RsrII sites may not be compatible.
For full activity, add fresh DTT.
BsiWI  (7706)
1 site
C G T A C G G C A T G C

BsiWI is typically used at 55°C, but is 50% active at 37°C.
BfuAI  (7639)
1 site
A C C T G C ( N ) 4 T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the BfuAI recognition sequence.
Sticky ends from different BfuAI sites may not be compatible.
BfuAI is typically used at 50°C, but is 50% active at 37°C.
BspMI  (7639)
1 site
A C C T G C ( N ) 4 T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the BspMI recognition sequence.
Sticky ends from different BspMI sites may not be compatible.
AgeI  (7240)
1 site
A C C G G T T G G C C A
EcoRI  (7120)
1 site
G A A T T C C T T A A G
PaeR7I  (7108)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
XhoI  (7108)
1 site
C T C G A G G A G C T C
BamHI  (7096)
1 site
G G A T C C C C T A G G

After cleavage, BamHI-HF™ (but not the original BamHI) can remain bound to DNA and alter its electrophoretic mobility.
PshAI  (7024)
1 site
G A C N N N N G T C C T G N N N N C A G

PshAI quickly loses activity at 37°C, but can be used at 25°C for long incubations.
BbsI  (6692)
1 site
G A A G A C N N C T T C T G N N ( N ) 4

Sticky ends from different BbsI sites may not be compatible.
BbsI gradually loses activity when stored at -20°C.
BglII  (1410)
1 site
A G A T C T T C T A G A
NcoI  (1421)
1 site
C C A T G G G G T A C C
PflMI  (1722)
1 site
C C A N N N N N T G G G G T N N N N N A C C

Sticky ends from different PflMI sites may not be compatible.
SgrDI  (1789)
1 site
C G T C G A C G G C A G C T G C
BsaBI  (2240)
1 site
G A T N N N N A T C C T A N N N N T A G
* Blocked by Dam methylation.
PsiI  (2540)
1 site
T T A T A A A A T A T T
MluI  (5256)
1 site
A C G C G T T G C G C A
dCas9
1423 .. 5526  =  4104 bp
1368 amino acids  =  158.3 kDa
   Segment 1:  
   1423 .. 1449  =  27 bp
   9 amino acids  =  1.1 kDa
Product: catalytically dead mutant of the Cas9 endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system
RNA-guided DNA-binding protein that lacks endonuclease activity due to the D10A mutation in the RuvC catalytic domain and the H840A mutation in the HNH catalytic domain
dCas9
1423 .. 5526  =  4104 bp
1368 amino acids  =  158.3 kDa
   Segment 2:  
   1450 .. 1452  =  3 bp
   1 amino acid  =  89.1 Da
Product: catalytically dead mutant of the Cas9 endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system
RNA-guided DNA-binding protein that lacks endonuclease activity due to the D10A mutation in the RuvC catalytic domain and the H840A mutation in the HNH catalytic domain
dCas9
1423 .. 5526  =  4104 bp
1368 amino acids  =  158.3 kDa
   Segment 3:  
   1453 .. 3939  =  2487 bp
   829 amino acids  =  96.3 kDa
Product: catalytically dead mutant of the Cas9 endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system
RNA-guided DNA-binding protein that lacks endonuclease activity due to the D10A mutation in the RuvC catalytic domain and the H840A mutation in the HNH catalytic domain
dCas9
1423 .. 5526  =  4104 bp
1368 amino acids  =  158.3 kDa
   Segment 4:  
   3940 .. 3942  =  3 bp
   1 amino acid  =  89.1 Da
Product: catalytically dead mutant of the Cas9 endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system
RNA-guided DNA-binding protein that lacks endonuclease activity due to the D10A mutation in the RuvC catalytic domain and the H840A mutation in the HNH catalytic domain
dCas9
1423 .. 5526  =  4104 bp
1368 amino acids  =  158.3 kDa
   Segment 5:  
   3943 .. 5526  =  1584 bp
   528 amino acids  =  60.9 kDa
Product: catalytically dead mutant of the Cas9 endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system
RNA-guided DNA-binding protein that lacks endonuclease activity due to the D10A mutation in the RuvC catalytic domain and the H840A mutation in the HNH catalytic domain
dCas9
1423 .. 5526  =  4104 bp
1368 amino acids  =  158.3 kDa
5 segments
Product: catalytically dead mutant of the Cas9 endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system
RNA-guided DNA-binding protein that lacks endonuclease activity due to the D10A mutation in the RuvC catalytic domain and the H840A mutation in the HNH catalytic domain
AmpR
10,193 .. 11,053  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   10,193 .. 10,984  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
10,193 .. 11,053  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   10,985 .. 11,053  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
10,193 .. 11,053  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
TagBFP
6400 .. 7095  =  696 bp
232 amino acids  =  26.2 kDa
Product: monomeric blue fluorescent protein
mammalian codon-optimized
TagBFP
6400 .. 7095  =  696 bp
232 amino acids  =  26.2 kDa
Product: monomeric blue fluorescent protein
mammalian codon-optimized
PuroR
7650 .. 8249  =  600 bp
199 amino acids  =  21.5 kDa
Product: puromycin N-acetyltransferase
confers resistance to puromycin
PuroR
7650 .. 8249  =  600 bp
199 amino acids  =  21.5 kDa
Product: puromycin N-acetyltransferase
confers resistance to puromycin
ori
9434 .. 10,022  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
9434 .. 10,022  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
5' LTR
11,986 .. 515  =  517 bp
5' long terminal repeat from murine embryonic stem cell virus
5' LTR
11,986 .. 515  =  517 bp
5' long terminal repeat from murine embryonic stem cell virus
3' LTR
8349 .. 8863  =  515 bp
3' long terminal repeat from murine embryonic stem cell virus
3' LTR
8349 .. 8863  =  515 bp
3' long terminal repeat from murine embryonic stem cell virus
PGK promoter
7130 .. 7629  =  500 bp
mouse phosphoglycerate kinase 1 promoter
PGK promoter
7130 .. 7629  =  500 bp
mouse phosphoglycerate kinase 1 promoter
gag (truncated)
987 .. 1403  =  417 bp
truncated MMLV gag gene lacking the start codon
gag (truncated)
987 .. 1403  =  417 bp
truncated MMLV gag gene lacking the start codon
RelA (p65) AD
6022 .. 6393  =  372 bp
124 amino acids  =  13.0 kDa
Product: transcriptional activation domain of human RelA, also known as p65 (O'Shea and Perkins, 2008)
RelA (p65) AD
6022 .. 6393  =  372 bp
124 amino acids  =  13.0 kDa
Product: transcriptional activation domain of human RelA, also known as p65 (O'Shea and Perkins, 2008)
MESV Ψ
579 .. 920  =  342 bp
packaging signal of murine embryonic stem cell virus
MESV Ψ
579 .. 920  =  342 bp
packaging signal of murine embryonic stem cell virus
AmpR promoter
11,054 .. 11,158  =  105 bp
AmpR promoter
11,054 .. 11,158  =  105 bp
SV40 NLS
5536 .. 5556  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T antigen
SV40 NLS
5536 .. 5556  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T antigen
SV40 NLS
5572 .. 5592  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T antigen
SV40 NLS
5572 .. 5592  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T antigen
ORF:  1423 .. 7104  =  5682 bp
ORF:  1893 amino acids  =  215.2 kDa
ORF:  5273 .. 5557  =  285 bp
ORF:  94 amino acids  =  10.0 kDa
ORF:  5819 .. 6148  =  330 bp
ORF:  109 amino acids  =  13.4 kDa
ORF:  7410 .. 8249  =  840 bp
ORF:  279 amino acids  =  30.1 kDa
ORF:  10,323 .. 10,589  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  11,439 .. 11,735  =  297 bp
ORF:  98 amino acids  =  11.7 kDa
ORF:  3336 .. 3704  =  369 bp
ORF:  122 amino acids  =  14.1 kDa
ORF:  6162 .. 6440  =  279 bp
ORF:  92 amino acids  =  9.4 kDa
ORF:  7575 .. 8300  =  726 bp
ORF:  241 amino acids  =  24.8 kDa
ORF:  362 .. 637  =  276 bp
ORF:  91 amino acids  =  9.9 kDa
ORF:  674 .. 934  =  261 bp
ORF:  86 amino acids  =  9.5 kDa
ORF:  10,193 .. 11,053  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
ORF:  3043 .. 3516  =  474 bp
ORF:  157 amino acids  =  17.4 kDa
ORF:  4828 .. 5214  =  387 bp
ORF:  128 amino acids  =  14.5 kDa
ORF:  6379 .. 6903  =  525 bp
ORF:  174 amino acids  =  17.8 kDa
ORF:  11,503 .. 11,733  =  231 bp
ORF:  76 amino acids  =  7.5 kDa
Try SnapGene and create your own beautiful maps

Individual Sequences & Maps

SnapGene offers the fastest and easiest way to plan, visualize, and document your molecular biology procedures.

Priced accessibly so that everyone in your lab can have a license.

Learn More...

SnapGene Viewer is a versatile tool for creating and sharing richly annotated sequence files. It opens many common file formats.

Free! Because there should be no barriers to seeing your data.

Learn More...

The map, notes, and annotations on this page and in the sequence/map file are copyrighted material. This material may be used without restriction by academic, nonprofit, and governmental entities, except that the source must be cited as "www.snapgene.com/resources". Commercial entities must contact GSL Biotech LLC for permission and terms of use.

Copyright © 2018 GSL Biotech LLC | Site Map | Privacy | Legal Disclaimers   Subscribe to Our Newsletter